Synthesis and antioxidant properties of an unnatural plasmalogen analogue bearing a trans O-vinyl ether linkage

Article abstract of DOI:10.1021/ol9009078

To assess the antioxidant behavior of trans-1, we first synthesized trans-allyl ether 4 by opening an (S)-glycildol derivative with an (E)-alk-2en-ol, and then produced the unnatural E-enol ether 1 by a stereoselective iridium(I)-catalyzed olefin isomerization. Natural cis-1 was preferentially degraded by HOCI and was more protective than trans-1 against lipid peroxidation Induced by a free-radical Initiator, demonstrating that the geometry of the 1-alkenyloxy bond participates In the antioxidant defensive role of 1.

Full text of DOI:10.1021/ol9009078

ORGANIC
LETTERS
2009
Vol. 11, No. 13
2784-2787
Synthesis and Antioxidant Properties of
an Unnatural Plasmalogen Analogue
Bearing a trans O-Vinyl Ether Linkage
Ravi S. Lankalapalli,† Joseph T. Eckelkamp,‡ Debajit Sircar,§ David A. Ford,‡
Papasani V. Subbaiah,§ and Robert Bittman*,†
Department of Chemistry and Biochemistry, Queens College of The City UniVersity of
New York, Flushing, New York 11367-1597, Department of Biochemistry and
Molecular Biology, St. Louis UniVersity School of Medicine, St. Louis, Missouri 63104,
and Department of Medicine, UniVersity of Illinois at Chicago, Chicago, Illinois 60612
robert.bittman@qc.cuny.edu
Received April 29, 2009
ABSTRACT
To assess the antioxidant behavior of trans-1, we first synthesized trans-allyl ether 4 by opening an (S)-glycidol derivative with an (E)-alk-2-
en-ol, and then produced the unnatural E-enol ether 1 by a stereoselective iridium(I)-catalyzed olefin isomerization. Natural cis-1 was preferentially
degraded by HOCl and was more protective than trans-1 against lipid peroxidation induced by a free-radical initiator, demonstrating that the
geometry of the 1′-alkenyloxy bond participates in the antioxidant defensive role of 1.
Plasmalogens are a subclass of glycerophospholipids that
bear an 1-O-alk-1′-(Z)-enyl chain at the sn-1 position of
glycerol (usual chain length, C16 or C18), sn-2 fatty acyl
chain (typically unsaturated or polyunsaturated), and a polar
headgroup at the sn-3 position (predominantly a mixture of
phosphoethanolamine and phosphocholine).1 Plasmalogens
comprise about 20% of the total phospholipids in humans;
they are especially abundant in human cardiac muscle,
skeletal muscle, and nervous tissues, which contain high
levels of polyunsaturated fatty acids.2 Since plasmalogen-
deficient cells are more susceptible to free radical mediated
oxidative damage than cells with normal plasmalogen levels,3
and because enol ethers are known to be susceptible to
oxidation,4 it has been proposed that the Z-enol ether
functionality, which is unique to plasmalogens, may serve
as a trap for reactive oxygen species. The vinyl ether linkage
is located in close proximity to the lipid-water interface,
and may thus protect the polyunsaturated chains of phos-
pholipids in membranes and lipoproteins from oxidation.5
The putative intermediates formed by [2 + 2] and [2 + 4]
(3) (a) Morand, O. H.; Zoeller, R. A.; Raetz, C. R. H. J. Biol. Chem.
1988, 263, 11597–11606. (b) Hoefler, G.; Paschke, E.; Hoefler, S.; Moser,
A. B.; Moser, H. W. J. Clin. InVest. 1991, 88, 1873–1879.
(4) For examples of 1,2-dioxetane formation from the reaction of enol
ethers with singlet oxygen, see: Gollnick, K.; Knutzen-Mies, K. J. Org.
Chem. 1991, 56, 4017–4027.
† CUNY.
‡ St. Louis University School of Medicine.
§ University of Illinois at Chicago.
(1) (a) Nagan, N.; Zoeller, R. A. Prog. Lipid Res. 2001, 40, 199–229.
(b) Brites, P.; Waterham, H. R.; Wanders, R. J. Biochim. Biophys. Acta
2004, 1636, 219–231.
(5) (a) Engelmann, B.; Bra¨utigam, C.; Thiery, J. Biochem. Biophys. Res.
Commun. 1994, 204, 1235–1242. (b) Reiss, D.; Beyer, K.; Engelmann, B.
Biochem. J. 1997, 323, 807–814. (c) Hahnel, D.; Beyer, K.; Engelmann,
B. Free Radical Biol. Med. 1999, 27, 1087–1094.
(2) Horrocks, L. A.; Sharma, M. In Phospholipids; Hawthorne, J. N.,
Ansell, G. B., Eds.; Elsevier: Amsterdam, 1982; pp 51-93.
10.1021/ol9009078 CCC: $40.75
Published on Web 06/05/2009
 2009 American Chemical Society

cycloaddition of singlet oxygen to this linkage are dioxetane
and ene intermediates, respectively, which subsequently
undergo degradation to generate a complex mixture of fatty
aldehydes (such as pentadecanal), 1-formyl-2-acyl-sn-glyc-
erophospholipids, and 1-(O-1′-hydroperoxy)-2-acyl-sn-glyc-
ero-3-phospholipids.6 Decomposition of the allylic hydro-
peroxides produces plasmalogen epoxides via a radical
process, and 1-formyl- and 1-lyso-phospholipids by hydro-
lytic processes.6d,7
Plasmalogens are more susceptible to oxidation than
phosphatidylcholine (PC) and sphingomyelin, the other major
membrane phospholipids;8 however, sphingomyelin has also
been found to inhibit PC peroxidation, albeit less efficiently
than plasmalogen, but its long-chain base contains a trans
double bond near the membrane-water interface.9a To
elucidate the role of the naturally occurring (Z)-O-vinyl
linkage of plasmalogen in protecting polyunsaturated lipids
from oxidative degradation, we describe herein the first
chemical synthesis of an unnatural analogue of plasmalogen
with an (E)-O-vinyl linkage at the sn-1 position (trans-1,
Figure 1)10 and a comparison of the antioxidant effects of
cis- and trans-1 in two model systems.
including the double bonds of lipids, to generate chlorinated
products.13 Recently, Davies and co-workers showed that
the kinetics of vinyl ether oxidation is several orders of
magnitude greater than that of aliphatic alkenes.14 The
products of plasmalogen oxidation by HOCl are 2-chloro
fatty aldehydes and 1-lysophosphatidylcholine (LPC).15
Phospholipid chlorohydrins in the sn-2 chain of unsaturated
LPC molecular species are also formed as secondary reaction
products by electrophilic attack of HOCl on alkenyl double
bonds.16 These chlorinated lipid species accumulate in
activated neutrophils, monocytes, ischemic myocardium, and
human atherosclerotic lesions and have potential roles in
many inflammatory disorders.17
Peroxidation of phospholipids containing a polyunsaturated
fatty acyl chain such as linolenic acid has been studied
frequently by using free-radical initiators, such as the water-
soluble thermolabile free-radical generator 2,2′-azobis(2-
amidinopropane) dihydrochloride (AAPH).18 As a model
system to compare the antioxidant capability of cis- and
trans-1, we studied the influence of 1 on the rate and extent
of peroxidation of 1-palmitoyl-2-linolenoylphosphatidylcho-
line (16:0-18:2-PC) in liposomes exposed to AAPH. The
reaction kinetics were monitored by measuring the formation
of conjugated diene lipid hydroperoxides, as monitored by
the absorbance at 234 nm.9 A234 is diminished when AAPH-
induced free-radical chain initiation in the polyunsaturated
acyl chain is suppressed.
Previous reports of the preparation of 1-O-alkenyl ether
derivatives of glycerol were based on elimination reactions
of R-halo cyclic glycerol acetals, which gave low yields and
poor stereoselectivity.19 To construct the glycerol backbone
with an O-1′-alkenyl chain at the sn-1 position, we used (S)-
glycidol and 1-hexadecanol as the starting materials for the
synthesis of trans-1 (see Supporting Information). As shown
in Scheme 1, the DPS ether of (S)-glycidol (3) was employed
in a regioselective BF3·OEt2-mediated ring-opening reaction20
of (E)-octadec-2-en-1-ol (2), prepared by DIBALH reduction
of ethyl (E)-octadecanoate. The expected attack at C-3
Figure 1. Structures of naturally occurring cis-1 and the unnatural
trans-1 analogue.
Hypochlorous acid is a highly reactive oxidant and
chlorinating agent that is produced endogenously when
physiological concentrations of chloride ion are oxidized by
the myeloperoxidase-catalyzed decomposition of hydrogen
peroxide.11 HOCl reacts with many biological molecules,12
(12) Pattison, D. I.; Davies, M. J. Curr. Med. Chem. 2006, 13, 3271–
3290.
(13) Albert, C. J.; Crowley, J. R.; Hsu, F. F.; Thukkani, A. K.; Ford,
D. A. J. Biol. Chem. 2001, 276, 23733–23741.
(14) Skaff, O.; Pattison, D. I.; Davies, M. J. Biochemistry 2008, 47,
8237–8245.
(15) Messner, M. C.; Albert, C. J.; Hsu, F. F.; Ford, D. A. Chem. Phys.
Lipids 2006, 144, 34–44. Primary amines react with HOCl to give
chloramines; therefore, we synthesized 1 with a phosphocholine instead of
a phosphoethanolamine head group to target the O-vinyl ether linkage in
the HOCl reaction and avoid having a competing reactive site.
(16) (a) Thukkani, A. K.; Albert, C. J.; Wildsmith, K. R.; Messner, M. C.;
Martinson, B. D.; Hsu, F. F.; Ford, D. A. J. Biol. Chem. 2003, 278, 36365–
36372. (b) Thukkani, A. K.; Hsu, F. F.; Crowley, J. R.; Wysolmerski, R. B.;
Albert, C. J.; Ford, D. A. J. Biol. Chem. 2002, 277, 3842–3849.
(17) (a) Thukkani, A. K.; Martinson, B. D.; Albert, C. J.; Vogler, G. A.;
Ford, D. A. Am. J. Physiol. Heart Circ. Physiol. 2005, 288, H2955–H2964.
(b) Thukkani, A. K.; McHowat, J.; Hsu, F. F.; Brennan, M. L.; Hazen,
S. L.; Ford, D. A. Circulation 2003, 108, 3128–3133. (c) Spickett, C. M.
Pharmacol Ther. 2007, 115, 400–409.
(6) (a) Zoeller, R. A.; Lake, A. C.; Nagan, N.; Gaposchkin, D. P.; Legner,
M. A.; Lieberthal, W. Biochem. J. 1999, 338, 769–776. (b) Zoeller, R. A.;
Morand, O. H.; Raetz, C. R. H. J. Biol. Chem. 1988, 263, 11590–11596.
(c) Stadelmann-Ingrand, S.; Favreliere, S.; Fauconneau, B.; Mauco, G.;
Tallineau, C. Free Radical Biol. Med. 2001, 31, 1263–1271. (d) Thompson,
D. H.; Inerowicz, H. D.; Grive, J.; Sarna, T. Photochem. Photobiol. 2003,
78, 323–330.
(7) Loidl-Stahlhofen, A.; Hannemann, K.; Felde, R.; Spiteller, G.
Biochem. J. 1995, 309, 807–812
(8) Hofer, G.; Lichtenberg, D.; Kostner, G. M.; Hermetter, A. Clin.
Biochem. 1996, 29, 445–450.
.
(9) (a) Subbaiah, P. V.; Sircar, D.; Lankalapalli, R. S.; Bittman, R. Arch.
Biochem. Biophys. 2009, 481, 72–79. (b) Recknagel, R. O.; Glende, E. A.
Jr. In Methods Enzymol., Vol. 105; Packer, L., Ed. Academic: San Diego
1984, pp. 331-337.
(18) Niki, E. In Methods Enzymol., Vol. 186; Packer, L., Glazer, A. N.,
Eds.; Academic: San Diego, 1990; pp 100-108.
(10) A semisynthetic route to racemic trans-1 via a lipase-catalyzed
hydrolysis of an 1-(alkenyloxy)-2,3-diacylglycerol was reported: Slotboom,
A. J.; De Haas, G. H.; van Deenen, L. L. M. Chem. Phys. Lipids 1967, 1,
192–208.
(19) For syntheses of mixtures of 1-O-(1′-alkenyl)glycerols and 2-O-
(1′-alkenyl)glycerols containing E/Z mixtures, see: (a) Piantadosi, C. F.;
Hirsch, A. F.; Yarbro, C. L.; Anderson, C. E. J. Org. Chem. 1963, 28,
2425–2428. (b) Craig, J. C.; Hamon, D. P. G. J. Org. Chem. 1965, 30,
4168–4175. (c) Otera, J.; Niibo, Y. Bull. Chem. Soc. Jpn. 1986, 59, 3977–
3978. (d) Pfaendler, H. R.; Mu¨ller, F. X. Synthesis 1992, 350–352.
(11) Senthilmohan, R.; Kettle, A. J. Arch. Biochem. Biophys. 2006, 445,
235–244.
Org. Lett., Vol. 11, No. 13, 2009
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in 96% yield.24 sec-Alcohol 5 was esterified with oleic acid,
using DCC in the presence of DMAP, affording ester 6. The
final steps in the synthesis of trans-1 required conditions that
did not cleave the acid-labile and oxidizable vinyl ether
moiety or the alkaline-labile acyl ester bond. After the silyl
ether in 6 was removed using TBAF (6 equiv) and imidazole
(2.5 equiv) in THF at -23 °C, the phosphocholine headgroup
was installed at the sn-3 position in 54% overall yield without
accompanying acyl migration by opening of a cyclic phos-
pholane intermediate in a pressure tube with anhydrous Me3N
(condensed at -10 °C) in MeCN/C6H6 (3:1) in the presence
of pyridine.25
Scheme 1. Preparation of Allyl Ether 4
The first model system we investigated was the reaction
of cis- and trans-1 with HOCl. The HOCl oxidation reaction
of cis- and trans-1 was analyzed using increasing concentra-
tions of HOCl (Figure 2). At both a 2- and 5-fold molar
provided an 8:1 (separable) mixture of allyl ether 4 and the
undesired regioisomer 4a.
Next, isomerization of the double bond was investigated
using transition metal complexes as catalysts.21 An initial
attempt using RhCl3·3H2O22 as a catalyst resulted in no
reaction. Fortunately, we found that use of (1,5-cycloocta-
diene)bis(methyldiphenylphosphine)iridium(I) hexafluoro-
phosphate (Ir(COD)[PMePh2]2PF6),23 after activation with
hydrogen for 5 min, resulted in the stereoselective isomer-
ization of allyl ether 4 to enol ether 5 in 2 h at rt, and with
the desired E double bond as the only product (Scheme 2);
Scheme 2
.
Ir(I)-Mediated Olefin Isomerization and Final
Conversion to trans-1
Figure 2. Disparate cis- and trans-1 oxidation by HOCl. The bars
show the amounts in nmol of 1 remaining (left panel) and 2-chlo-
rooctadecanal produced (right panel) after treatment with various
HOCl/1 molar ratios for 5 min at 37 °C. Data are the mean ( SD of
3 experiments. See Supporting Information for experimental details.
ratio of [HOCl]/[1], the cis-vinyl ether linkage was oxidized
faster than the trans linkage. This selectivity was not observed
(21) (a) Krompiec, S.; Kuz´nik, N.; Urbala, M.; Rzepa, J. J. Mol. Catal.
A: Chem. 2006, 248, 198–209. (b) Kuz´nik, N.; Krompiec, S. Coord. Chem.
ReV. 2007, 251, 222–233.
(22) Grieco, P. A.; Nishizawa, M.; Marinovic, N.; Ehmann, W. J. J. Am.
Chem. Soc. 1976, 98, 7102–7104.
(23) (a) Oltvoort, J. J.; Van Boeckel, C. A. A.; De Koning, J. H.; Van
Boom, J. H. Synthesis 1981, 305–308. (b) Westcott, S. A. In e-EROS
Encyclopedia of Reagents for Organic Synthesis; Wiley: Chichester, UK,
2001.
1
JH1′-H2′ ) 12.6 Hz at δ 6.24 ppm in the H NMR spectrum
of 5 is indicative of the desired E isomer, which was obtained
(24) The Z isomer of 5 was prepared by Lindlar reduction of an
O-alkynyl ether; for the high-field doublet of the cis-O-vinyl proton JH1′-H2′
) 6.20 Hz at δ 5.94 ppm.25
(20) For BF3•OEt2-mediated opening of glycidyl derivatives with
saturated aliphatic alcohols, see: (a) Guivisdalsky, P. N.; Bittman, R. J. Am.
Chem. Soc. 1989, 111, 3077–3079. (b) Guivisdalsky, P. N.; Bittman, R. J.
Org. Chem. 1989, 54, 4637–4642.
(25) Qin, D. H.; Byun, H. S.; Bittman, R. J. Am. Chem. Soc. 1999, 121,
662–668.
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at a higher [HOCl]/[1] molar ratio, probably because
competing reactions that consume HOCl take place, such as
addition to the double bond in the sn-2 fatty acyl chain of
1.17c
oxidation by 33% vs only 12% inhibition by trans-1, based
on the slopes of the reaction curves in the linear range
(0-100 min; Figure 4). Similar data were obtained when 1
ESI-MS analyses of the reaction mixture with 1 mM HOCl
demonstrated that the other product of HOCl oxidation is
LPC containing an oleic acid moiety (m/z 544.24) (Figure
3). The chlorohydrin of this material, formed by reaction
with HOCl,15 is a byproduct with m/z 596.18.
Figure 4. Effect of cis- and trans-1 on the oxidation of 16:0-18:2-PC
induced by AAPH.
mM AAPH was used (data not shown). We also determined
the effect of cis- and trans-1 on 16:0-18:2-PC oxidation in
liposomes in the presence of 50 µM Cu2+ as the oxidizing
agent, which facilitates free-radical chain propagation.29 We
found that trans-1 was completely ineffective in protecting
16:0-18:2-PC oxidation whereas cis-1 inhibited PC oxida-
tion by 45% (results not shown).
In summary, the first chemical synthesis of trans-1, an
unnatural analogue of plasmalogen bearing a trans O-vinyl
linkage at the sn-1 position, has been achieved. A key step
involves E stereoselective enol ether formation of 5 via an
iridium(I)-mediated olefin isomerization of O-allyl ether 4.
The data shown in Figures 2 and 4 indicate that the geometry
of the alkenyl ether linkage of plasmalogen plays a significant
role in plasmalogen’s antioxidant properties.
Figure 3. ESI-MS of the HOCl oxidation products. cis- or trans-1
(50 nmol; (M + Na)+, m/z 794.35) was incubated in 500 µL of 20
mM phosphate-buffered saline containing 0.1 mM diethylenetri-
aminepentaacetic acid (pH 7.0) in the presence of 1 mM chloride-
free NaOCl for 5 min at 37 °C.26
Plasmalogens are known to protect membrane phos-
pholipids against radical-induced damage.1a Therefore, we
next evaluated the ability of cis- and trans-1 to protect
16:0-18:2-PC from AAPH (3.3 mM) induced peroxidation
in liposomes prepared with 80 mol % 16:0-18:2-PC and
20 mol % cis- or trans-1. The oxidation of 16:0-18:2-PC
Acknowledgment. This work was supported in part by
NIH grants HL083187 (R.B.), HL088072 and HL074214
(D.A.F.), and HL68585 and DK078165 (P.V.S.).
was monitored by the increase in A234,
27 a measure of con-
Supporting Information Available: Experimental pro-
cedures and NMR spectra for the reported compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
jugated diene hydroperoxide formation in the sn-2 fatty acyl
group of PC.28 cis-1 inhibited the rate of 16:0-18:2-PC
(26) The reactions were terminated by addition of MeOH, the products
were extracted into CHCl3, resuspended in 500 µL of MeOH/CHCl3 (4:1)
containing 1 µM NaOH, and analyzed by ESI-MS (flow rate, 3 µL/min).
Samples were analyzed in the positive ion mode with detection of their
sodiated adducts.
OL9009078
(27) To correct for changes in A234 arising from AAPH-induced
plasmalogen oxidation, liposomes were prepared with only cis- or trans-1
at the same concentrations present in the 16:0-18:2-PC + 1 liposomes.
(28) Esterbauer, H.; Striegl, G.; Puhl, H.; Rotheneder, M. Free Radical
Res. Commun. 1989, 6, 67–75.
(29) Burkitt, M. J. Arch. Biochem. Biophys. 2001, 394, 117–135.
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